Yersinia pestis (Yp), the causative agent of the plague (PL), and Mycobacterium tuberculosis (Mt), the etiologic agent of tuberculosis (TB), are pathogens with an important impact in both global and national public health. The Center for Disease Control and Prevention (CDC) has included Yp and multiple-drug resistant (MDR) Mt in categories A and C, respectively, of biological agents for public health preparedness against bioterrorism. The lack of suitable antibiotics to treat outbreaks of MDR PL and MDR TB resulting from natural emergence or bioterrorism is an alarming scenario. Counter-bioterrorism measures require the development of an arsenal of new antimicrobial drugs against conventional and unconventional targets in Yp and Mt. Most antibiotics in clinical use to treat TB target enzymes involved in protein, nucleic-acid, or cell-wall component synthesis, whereas PL is primarily treated with antibiotics that inhibit protein or DMA synthesis. Many lines of evidence indicate that iron acquisition systems based on iron-chelators, referred to as siderophores, are required for the virulence of these pathogens. In particular, Yp siderophore biosynthesis is one of the targets for the development of drugs for intervention and treatment of Yp infections indicated in the Counter-Bioterrorism Research Agenda of the NIAID for CDC Category A Agents. Organism-specific drugs that inhibit siderophore biosynthesis will have use as therapies, alone or in combination with other drugs, to treat infections with MDR Yp and Mt strains. The goal of this proposal is to find compounds that inhibit Yp and Mt siderophore biosynthesis. To achieve this goal, we will screen combinatorial compound libraries for inhibitors of selected siderophore biosynthesis enzymes and synthesized rationally designed mechanism-based inhibitors of such enzymes. The selected enzymes have no homologs in humans. The identified inhibitors will be characterized in cell free assays and evaluated as inhibitors of bacterial growth, siderophore biosynthesis, and iron uptake. The identified inhibitors, and the chemoinformation generated by their analysis, will constitute a base for the rational development of improved inhibitors of siderophore-mediated iron acquisition in Yp and Mt. These inhibitors represent a first step towards developing antimicrobials targeting such process. These antimicrobials will constitute a base for subsequent studies for the development of new drugs that, alone or in combination therapies, are anticipated to be useful in the treatment of MDR Yp and Mb infections.